How can Blood Stabilizers be used for Validation of New Instruments?
Quality Pathology is essential for about 60% of all diagnoses. Thus, quality of pathology services is critical for good care and management. In health care there are increasing numbers of methods we can use to monitor quality: Audit (internal and external) Staff Training Internal Quality Control Standards (control or reference material) External Quality Control Unknown samples (peer to peer comparison, method means, group comparisons)
Quality One of the key components to enable the monitoring of quality is: Standardisation
Standardisation Quality serves as a basis or example or principle to which others conform or should conform or by which the accuracy of others is judged. Oxford Dictionary
Eurostandards What was it? Why did we do it? Who did it involve? What did it achieve?
Eurostandards What was it? What was it? EC Quality of Life and Management of Living Resources Proposal (Fifth Framework) Area Support for Research Infrastructure Designed to create: Central facility for the production of stabilised reference standards and external quality assessment in clinical flow cytometry 3 year funded (2.8 million Euros) project started 1 st January 2001
Eurostandards Why do it? Why do it? Use of flow cytometry in the clinical arena has increased dramatically in the last 15 years. Currently, a conservative estimate puts the number of flow cytometers in clinical use at over 5,000 in European establishments. The European cost for providing such a service is estimated to be 2000 million Euros per annum. Interestingly in the UK the average amount spent on EQA is 390 Euros per annum (Equivalent to <0.3% of the facilities annual budget). Better standardisation could reduce the estimated expenditure on repeat testing by as much as 300 million Euros.
Eurostandards Who did it involve? It involves 11 sites in 8 countries. Sheffield, Rotterdam, Urbino, Regensburg, London, Salamanca, Milan, Stockholm, Lisbon, Athens, Potters Bar Sheffield coordinating centre Group met twice per annum and communicated with regular telephone conference calls & e-mail
Eurostandards What have we achieved?
Eurostandards What did it achieve? Proof of Principle Establish European & National EQA programmes.
Summary of degradation studies Approximately 15-18months shelf life (depending on specimen type). Will need to use appearance and general feel of material as an indication of shelf life, this however varies between materials and can be subjective. Ideally need to send samples out: soon after filling, and then at 6-8 monthly intervals for evaluation, in order for 3 or 4 assays to be completed within 18 months to have large scale data on sample degradation and have agreement on shelf life.
Work package 1 02/ 214 CD4 counts l u / 900 t n800 u o700 c 600 l 500 l e400 c 300 200 100 0 Box Plot Grouping Variable(s): PARTNER Split By: DAY Inclusion criteria: Criteria 6 from 02214stats.svd 1 p 2 p 3 p 4 p 5 p 6 p 7 p 8 p 9 p 0 1 p 1 1 p day 0 day 4 Partner 1 and partner 4 had significant difference between days. Overall mean = 481 cells/ul Overall CV = 11.7%
UK NEQAS/IQA Partnership
UKNEQAS / IQA International Partnership UKNEQAS Team Provides QA/QC Send out to NIH Supported Sites IQA Team Monitors Site Performance and Develops Site Specific Training
UK NEQAS/IQA Initiative Panel of stabilised samples devised to examine the individual laboratories ability to measure CD4 + T lymphocytes Involves a panel of 20 samples with CD4 ranges from 80 - >800 CD4 + cells/µl The technology confidence limits are now known and individual performance can be aligned to what is expected. Qualifying round of samples before embarking on EQA During course of EQA centres monitored and if show out of consensus results will be able to re-run specimens to determine problem area
UKNEQAS / IQA International Partnership Veritas Latin for Truth Verification Panel Quality Assessment VERIQAS
UK NEQAS/IQA Initiative Qualifying panel with known defined limits IQA Participant Pass Monitor Fail UK NEQAS EQA Participation Improved Clinical Trials and Lab Practice
VERIQAS Panel Layout 20 samples single samples replicate samples Assessment Protocol Ability to meet QA sample target zone (e.g. 150 CD4 cells) Whole blood with CD4 absolute range engineered ~ 80 to 850 cells/ul Reproducibility target zone (e.g., CV of replicates)
1000 Validation Panel CD4 Results Donor A Target CD4 > 350 900 800 700 CD4 ABS 600 500 400 300 Dual Platform Mean = 819 +/- 20 5 th = 788 95 th = 850 FACSCount Truecount Mean = 779 +/- 37 5 th = 734 95 th = 846 200 100 0 1 Mean = 797 +/- 29 5 th = 758 95 th = 843 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 UK N = NEQAS 33 for Leucocyte Immunophenotyping
UKNEQAS + IQA Data Set Mean Minimum Maximum SD +2SD -2SD Sample A 823 724 983 56 936 710 Sample B 92 70 108 10 112 73 Sample C 109 79 136 10 130 88 Sample D 108 94 133 9 127 90 Sample E 189 147 233 15 219 159 Sample F 231 197 280 16 263 198 Sample G 245 210 269 12 269 221 Sample H 269 229 327 29 326 212 Sample I 308 262 364 29 365 250
VEIQAS Panel E CD4 Count 300 250 200 150 100 50 0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 Replicate CD4# UK NEQAS CD4# IQA Mean +/- 2SD
Comparison of Absolute CD4 Counts in Two Centres (Cells/ul) 1000 800 R 2 = 0.9938 IQA 600 400 200 0 0 200 400 600 800 1000 1200 UK NEQAS
Comparison of Absolute CD4 Counts in Two Centres (Cells/ul) IQA 350 300 250 200 150 100 50 0 R 2 = 0.9621 0 50 100 150 200 250 300 350 400 UK NEQAS
Technology Confidence Limits and how can we use them?
Relative Error for CD4+ T cells (results based on 48 send outs to 387 laboratories) Graph 2 - Relative Errors for CD3+/CD4+ Counts Using Differing Technologies Relative Error (%) 35 30 25 20 15 Relative error(%) - Kunkl Relative error(%) - trucont Relative error(%) -flowcount Relative error (%) - dual 10 5 0 0.047 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.913 CD4 count (Cells per microlitre)
Relative Error for CD4+ T cells (results based on 48 send outs to 387 laboratories) Figure 1-99.9% Confidence Limits for CD3+/CD4+ Lymphocyte Enumeration by Single and Dual Platform Technologies. 2200 2000 1800 1600 1400 Dual Platform Confidence Limits Flow Count Confidence Limits TruCOUNT Confidence Limits 99.9 CL (cells/!l) 1200 1000 800 600 400 200 0 0 200 400 600 800 1000 1200 1400 1600 1800 2000 CD3+/CD4+ cells/!l
99.9% confidence limits for CD3+/CD4+ counts at a level of 500 cells/µl Method Lower 99.9% Confidence Limit Upper 99.9% Confidence Limit Confidence Limit Range Dual Platform 425 580 155 Flow Count (Single platform) 425 530 105 TruCOUNT (Single platform) 480 540 60
Sample Stabilizers Transportation introduces two important factors: Time & Sample Integrity Several haematology and immunology guidelines have published specific time frames for time to completion of analysis. Increased burden on the staff, budget and courier costs
Cyto-Chex Advantages Preserves light scatter characteristics Simple to use Up to 10 days sample preservation Allows whole blood lysis Disadvantages Dilution factor (1:1) Antigen susceptibilty Cannot be used on haematology analysers Cannot use on single platform Cannot extract DNA/RNA
TransFix Termed TRANSFix because it allows TRANSportation of FIXed Specimens. Minimal dilution effects (10ul per 1ml Sample). Can be used on normal, leukaemia and HIV patients. Facilitates haematological analysis. Minimal effect on antigen density. Samples stable for up to 10 days *. All samples kept at 4 o C and allowed to warm to R.T. (approx. 1 hour) before use. Facilitates flow cytometric analysis. Can be added immediately after draw or produced in an anticoagulated form.
TransFix Haematology Analysis 16 14 12 10 8 6 4 2 0 Day 1 Day 3 Day 5 Day 7 WCC Hb RBC 16 14 12 10 8 6 4 2 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 WCC Hb RBC Coulter VCS Sysmex NE1500 Analysis
TransFix Flow Cytometric Analysis Preservation of Lymphocyte Subsets When Using TRANSFix 80.00 70.00 Normal s Unfixed TransFix Specimens CD 3 Percentage of Lymphocytes 60.00 50.00 40.00 30.00 20.00 10.00 CD 4 CD 8 CD 16/56 CD 19 0.00 Day 1 Day 2 Day 3 Day 4 Day 6 TIME
TransFix Flow Cytometric Analysis 800 Effect of TransFix and Cytochex on CD4 Median Channel Fluorescence Median Channel 750 700 650 600 550 500 450 400 Day1 Day2 Day3 Day4 Day6 Time TRANSFIX CD4 NORMAL CD4 CYTOCHEX CD4
Conclusions Disadvantages Stabilised material not suitable for manual technologies such as Dynabeads. Not suitable for obtaining haematology differentials. Scatter can be a little confusing for new laboratories.
Conclusions Advantages Stabilised material offer a superior alternative than fresh blood to validate different single platform instrument technologies. Longitudinal studies easily conducted. Individual laboratory performance easily monitored. Can be used for teaching, training and troubleshooting. Samples can be engineered to have varying lymphocyte subset counts. Do not have to be HIV+
TransFix Publications Evaluation of leukocyte stabilisation in TransFix R -treated blood samples by flow cytometry and transmission electron microscopy. Canonico B, et al (2004) J Immunol Methods. 29:67-78 CD45 assisted PanLeucoGating for Accurate, Cost Effective Dual Platform CD4+ T cell enumeration. Glencross, D et al (2002) Cytometry (Communications in Cytometry) 50:69-77 Affordable CD4+ T cell counts by flow cytometry. II. The use of fixed whole blood in Resource-poor Settings. Jani I., et al (2001) Journal of Immunological Methods 257:145-154 Evaluation of TransFix, a commercial whole blood stabilizing reagent. This product reduces HIV replication. Kim et al: Cytometry Clinical Cytometry (2002) 50: 281 Evaluation of stabilised blood cell products as candidate preparations for quality assessment programs for CD4 T-cell counting. M. Bergeron, A.Shafaie, T.Ding, S.Phaneuf, N.Soucy, F. Mandy, J.Bradley & J.Fahey Cytometry (2002) 50, 86-91